This is in the general area of protein receptors, and more specifically relates to isolation and cloning of a D1-like receptor activity modifying protein (RAMP) which is a single transmembrane protein, designated P24, or calcyon, that interacts with a C-terminal intracellular segment of the hD1 DA receptor, and use thereof in screening for therapeutics and diagnostics.
The human D1 dopamine (DA) receptor displays micromolar affinity for the neuromodulator DA. Currently, the management of many psychiatric and movement disorders relies heavily on the inhibition or facilitation of DA at its receptors. Via DA receptors, DA and DA mimetic ligands, such as antipsychotic drugs, exert both short and long term changes in ion channel activity, protein kinase/phosphatase activities, and gene expression (Artalejo et al., Nature 348:239-42 (1990); Steiner and Gerfen, J. Comp. Neurol. 353:200-12 (1995); Surmeirer et al., Neuton 14:385-97 (1995)). The design of most of these drugs has been based on a xe2x80x9cD1xe2x80x9d and xe2x80x9cD2xe2x80x9d DA receptor subtype paradigm. flowever, molecular cloning has led to the identification of five mammalian DA receptor subtypes (D1-D5) (reviewed in Gingrich and Caron, Annu. Rev. Neurosci. 16:299-321 (1993)). Individual subtypes have been further characterized as D1-like (D1 and D5) or D2-like (D2, D3 and D4) based on their selectivity for either classical xe2x80x9cD1xe2x80x9d or xe2x80x9cD2xe2x80x9d dopaminergic ligands. Thus, conceptualizations of how DA modulates the mesolimbic, mesocortical and nigrastriatal pathways through xe2x80x9cD1xe2x80x9d and xe2x80x9cD2xe2x80x9d receptors are inadequate given the added molecular complexity of the DA receptor system. The functional implications of these xe2x80x9cnewxe2x80x9d xe2x80x9cD1xe2x80x9d and xe2x80x9cD2xe2x80x9d DA receptor subtypes for DA in regulating cognitive, motor and associative functions is currently unknown. However, their discovery presents new opportunities for obtaining a more precise understanding of the role of dopaminergic neurotransmission in these processes. In addition, a complete understanding of the cellular and molecular processes regulating the specific subtype functions is crucial for dealing with disorders like schizophrenia, Parkinson""s disease, Tourette""s syndrome, and drug addiction that appear to involve dysfunction in the DA system.
In brain, D1 receptors are most abundant in the caudate nucleus, where they are involved in the control of movement. D1 receptors are also found in prefrontal cortex (PFC) where they are required for working memory, a form of memory impaired in schizophrenia. In PFC, D1 receptors are present in pyramidal cell dendritic spines typically located several micrometers away from DA terminals.
Immunohistochemical analyses of the D1-like dopamine (DA) receptor subtypes, D1 and D5, shows that each receptor protein has a unique cellular and subcellular distribution within the mesocortical, mesolimbic, and nigrastriatal pathways. These results support the notion that each D1-like subtype serves a distinct function. However, the molecules that may mediate subtype-specific signal transduction differences in vivo have not yet been identified. In addition, details regarding the processes specifying the subcellular distribution of each receptor subtype are unclear. Without this molecular information, it is difficult to understand the physiologic requirements for multiple D1-like subtypes. Examination of well-characterized systems indicates that most processes in cells are mediated by protein complexes created by specific protein-protein interactions (Formnosa et al., In: Methods in Enzymology: Academic Press, Inc. pp 24-45 (1991)).
As a group, the five mammalian DA receptor subtypes comprise a subfamily of the G-protein coupled receptor (GPCR) superfamily with predicted seven transmembrane topology. Similarities in genomic organization, sequence, and G-protein coupling suggest that the two DA receptor families evolved from prototypical D1 and D2 receptor genes via duplication and divergence (O""Dowd et al., in Handbook of Receptors and Channels, CRC Press, Inc., pp. 95-123 (1994)). Although each of the D1-like and D2-like subtypes are presumed to serve distinct functions, subtype-specific signal transduction differences have not been identified in vivo. Without this functional information. it is difficult to understand the physiologic requirements for multiple D1-like and D2-like receptor subtypes. Using the D1 and D5 D1-like subtypes as a model, one can elucidate the physiologic basis for multiple DA receptor subtypes by defining molecular determinants of their functions. Information obtained from this research should lead to a more sophisticated understanding of the principles guiding the functional organization of dopaminergic pathways. Several classes of proteins that regulate GPCR""s are known, yet none have been found to alter the sensitivity of D1 receptors in vivo.
There is ample pharmacological, electrophysiological and behavioral evidence to testify to the importance of D1-like receptors in cognitive and motor processes under normal or pathological conditions, including tardive dyskinesia (Ellison and See, Pyschopharmacology 98:564 (1989); Spooren et al., Euro. J. Pharmacol. 204:217 (1991)), Parkinson""s Disease (Gilmore et al., Neuropharmacology 34:481-8 (1995)), working memory (Sawaguchi and Goldman-Rakic, Science 251:947-50 (1991)), and long term potentiation (Huang and Kandel, Proc. Natl. Acad. Sci. USA 92: 2446-50 (1995)). However, whether the primary D1-like receptor involved is D1, or D5, or both, in each of these processes/behaviors is completely unclear because both subtypes have similar affinities for xe2x80x9cD1xe2x80x9d receptor agonists and antagonists. mRNA and protein localization studies in rodent and primate have provided the most revealing insights into the different functions of the D1 and D5 subtypes in vivo (Huntlcy et al., Mol. Brain Res. 15:181-8 (1992); Levey et al., Proc. Natl. Acad. Sci. USA 90:8861-5 (1993); Smiley et al., Proc. Natl. Acad. Sci. USA 91:5720-4 (1994); Laurier et al., Mol. Brain Res. 25:344-350 (1994); Bergson et al., J. Neurosci. 15:7821-36 (1995)). Although the D1 subtype is the most abundant DA receptor subtype, many aspects of the D5 subtype""s localization in cerebral cortex and limbic nuclei suggest that it may support DA""s actions in the higher cognitive, associative and affective processes uniquely associated with humans. In contrast, the most abundant expression of D1 receptors is detected in the basal ganglia nuclei which are pimarily associated with movement.
Previous studies carried out with subtype-specific antibodies indicate that D1 and D5 receptor proteins are typically coexpressed in pyramidal neurons of monkey prefrontal cortex. However, initial electron microscopic studies suggest that D1 receptors are preferentially localized in spines of pyramidal neurons, and D5 receptors are mainly associated with their apical dendrites (Bcrgson et al., J. Neurosci. 15:7821-36 (1995)). As the synaptic input to spines is excitatory, and synaptic input to shafts is generally inhibitory (Jones, Cerebral Cortex 3:361-72 (1993); Harris and Kater, Annu. Rev. Neurosci. 17:341-71 (1994); Smith et al., J. Comp. Neurol. 344:1-19 (1994)), it seems reasonable to speculate that the two D1-like receptors may, in fact, be carrying out different functions. Their differential localization in pyramidal cell dendritic spines and shafts is consistent with the idea that D1 and D5 receptors initiate biochemical events that modulate excitatory or inhibitory synaptic transmission, respectively. This possibility has been supported by numerous electrophysiological studies (Cepeda et al., Proc. Natl. Acad. Sci. USA 90:9576-80 (1993); Cameron and Williams, Nature 366-344-7 (1993); Taber and Fibiger, J. Neurosci. 15:3896-904 (1995)). Indeed, a recent electrophysiological study demonstrated that DA""s normal ability to potentiate responses to NMDA is blunted in D1 knockout mice (Levine et al., J. Neurosci. 16:5870-82 (1996)).
D1 and D5 receptors, like a number of other GPCRs expressed in brain, stimulate adenyl cyclase in the presence of agonist presumably via coupling to a Gs-like G-protein. However, knowledge of whether D1 or D5 subtypes elicit specific physiological responses in vivo is lacking. It is also unclear whether receptor-specific regulatory steps exist to modify D1 versus D5 receptor activation. This molecular information is critical for developing therapies that might inhibit or activate a D1 or D5 specific function.
It is an object of the present invention to provide reagents which can be used to identify determinants which may permit the D1 and D5 subtypes to elicit unique cellular responses.
A number of cDNA clones whose products may interact with D1 receptors in vivo were identified. One of the cDNA clones, K 37, was characterized further. The protein (P24) encoded by K 37 is localized in dendrites and spines of pyramidal cells in PFC. The extent of overlap between P24 expressing and D1 receptor expressing pyramidal cells appeared to be 100%. In contrast, only a limited number of D1 receptor antibody labeled neurons in caudate expressed P24. P24 lowers the threshold of D1 receptor response to dopamine (DA) by an order of magnitude. Sequence similarity suggests P24 is a diverged member of the RAMP family. The P24 protein is therefore referred to as a D1 DA RAMP, and was given the name Calcyon. The isolated protein and nucleotide molecule encoding the protein, as well as primers for the nucleotide, are described. The protein and compounds modifying DA binding to the receptor or calcium release which is mediated by Calcyon are useful in research studies, drug screening, and therapeutically.